Two allelic variants and eight site-directed mutants of cytochrome P450 2B1 differing at residue 478 have been expressed in COS cells and assayed for androstenedione hydroxylase activities. The 478Gly and 478Ala variants and five mutants (Ser, Thr, Val, Ile, and Leu) exhibited 16beta-OH:16alpha-OH ratios ranging from 0.7 to 9.3, whereas the Pro, Glu, and Arg mutants were expressed but inactive. The seven samples active toward androstenedione also exhibited testosterone 16beta-OH:16alpha-OH ratios ranging from 0.4 to 2.3. With both steroids, the Gly variant had the highest 16beta-hydroxylase activity, and the 16beta-OH:16alpha-OH ratio increased with the size of aliphatic size chains (Ala, Val, and Ile/Leu). The highest ratio of androgen 15alpha:16-hydroxylation was observed with the Ser mutant. On the basis of previous work indicating decreased susceptibility of the 478Ala variant in liver microsomal and reconstituted systems to inactivation by chloramphenicol analogs, methodology was refined for monitoring enzyme inactivation in COS cell microsomes. The Gly and Ala variants were inactivated by chloramphenicol with similar rate constants, whereas the Ser and Val mutants were inactivated more slowly, and the Leu mutant was refractory. Only the Gly variant was inactivated by the chloramphenicol analog N-(2-p-nitrophenethyl)chlorofluoroacetamide. Thus, the side chain of residue 478 appears to be a major determinant of enzyme inactivation as well as of androgen hydroxylation. Overall, this study demonstrates the importance of the amino acid at position 478 in dictating the substrate specificity of P450 2B1 and provides firm experimental evidence for a model in which this residue comprises part of a substrate recognition site.